In order to provide desired preventative or diagnostic health care, a physician must often determine the level of various analytes in a patient's blood, urine or other body fluids. For example, the level of glucose is often important in the diagnosis and subsequent treatment of diabetes. The level of hemoglobin in the blood is often important for effective diagnosis and treatment of anemia or other related blood abnormalities.
Another important analyte which physicians often monitor is bilirubin. Bilirubin is a degradation product of hemoglobin. Approximately 200 to 230 mg of bilirubin and its derivatives are formed each day in the normal human adult. As part of normal human metabolic processes, the major portion of this daily bilirubin production is excreted or degraded into other derivatives.
Excessive amounts of bilirubin occur within the human body through overproduction of bilirubin as in the case of excessive hemolysis or by retention of bilirubin due, for example, to liver failure. The result of excessive bilirubin within the human body is jaundice. Jaundice is characterized by markedly elevated serum bilirubin levels, for example, 10 mg of bilirubin per dL of serum or higher compared with the normal adult range of 0.1 to about 1 mg of bilirubin per dL of serum. There is increasing evidence that excessive amounts of bilirubin in the blood lead to an undesirable increase in bilirubin concentration within body cells which interferes with various cellular processes. Given this background, the clinical diagnostic significance of bilirubin, in tests for liver and other related organ functions, is self evident.
Perhaps the most widely used assay for bilirubin has been the so called diazo method. In this method, a sample of liquid suspected of containing bilirubin is contacted with a reagent composition which includes a diazonium salt. The diazonium salt reacts with bilirubin to form two azobilirubin fragments. The azobilirubin has an extinction coefficient which is higher than that of bilirubin itself and is easily detectable.
Many diazonium salts have been suggested for use in the diazo method for determining bilirubin. For example, certain 2,4- and 2,5-phenyldiazonium salts (e.g. 2,4- and 2,5-dichlorophenyldiazonium salts) and diazotized sulfanilamide have been used for the detection of bilirubin in serum and urine. However, methods using these diazonium salts are known to be relatively insensitive. Further, some of these diazonium salts, when dry, are explosively unstable, i.e. subject to shock induced decomposition. Thus, handling of these compounds in bilirubin assays, and particularly dry assays, is quite hazardous.
Certain substituted sulfanilamide and carbonamide diazonium salts which are less prone to shock induced decomposition have been found useful in bilirubin assays. These salts and assays are the subject of U.S. Pat. No. 4,468,467 (issued Aug. 28, 1984 to Babb et al). Those salts and assays represent a significant improvement in the clinical chemistry art, overcoming the shortcomings of previously-known bilirubin assays. This improved assay is also described by Babb and co-authors in Clin. Chem., 29(1), pp. 37-41 (1983).
Many substances, both foreign and native, are present in biological fluids, which substances cause serious interferences in the quantitative analyses of analytes, including bilirubin. Notwithstanding the significant improvement provided by the invention of Babb and Dappen noted hereinabove, there was a need prior to the instant invention to provide further improvements in the bilirubin assay. For example, with a small percentage of patient serum samples, e.g. those obtained from hemodialysis or other renal-defective patients, interferences were observed to be influential in the end result, detracting from assay accuracy. It is desirable to remove such interferences, thereby providing an assay that is highly accurate with all patient samples including samples obtained from patients having kidney problems.
Known procedures for eliminating interferences in assays include sample pretreatment, sample blanking and polychromatic (i.e. multiple wavelength) analyses. Each of these procedures, however, has its disadvantages. Sample pretreatment is a tedious and imprecise operation and is not readily adaptable to dry chemistry assays. Sample blanking doubles the effort, reagent amount and cost of each assay while being ineffective with regard to interferents formed in situ during the assay. The known polychromatic analysis requires pure standards and knowledge of the exact molecular identity or concentrations of predetermined interferents. See, e.g. Hahn et al, Clin. Chem., 25(6), pp. 951-959 (1979). Again, this technique would not be useful where the interferent is unknown and cannot be determined prior to the assay.
None of these known procedures has proved effective for eliminating the observed interference in the bilirubin assay described and claimed in U.S. Pat. No. 4,468,467 noted hereinabove. Neither the identity of the interferent nor its concentration (which can vary from sample to sample) is known. It is likely that the interferent is formed during the assay. This precludes use of known techniques for removing the effects of interferents.
Therefore, there is a need in the art for a means for overcoming the effect of undefined interferents, such as those formed in situ, i.e. during the analysis. In particular, there is a need for a means whereby assay accuracy for all patient samples, not just some samples, is achieved.